NOrMAL: accurate nucleosome positioning using a modified Gaussian mixture model

Motivation: Nucleosomes are the basic elements of chromatin structure. They control the packaging of DNA and play a critical role in gene regulation by allowing physical access to transcription factors. The advent of second-generation sequencing has enabled landmark genome-wide studies of nucleosome positions for several model organisms. Current methods to determine nucleosome positioning first compute an occupancy coverage profile by mapping nucleosome-enriched sequenced reads to a reference genome; then, nucleosomes are placed according to the peaks of the coverage profile. These methods are quite accurate on placing isolated nucleosomes, but they do not properly handle more complex configurations. Also, they can only provide the positions of nucleosomes and their occupancy level, whereas it is very beneficial to supply molecular biologists additional information about nucleosomes like the probability of placement, the size of DNA fragments enriched for nucleosomes and/or whether nucleosomes are well positioned or ‘fuzzy’ in the sequenced cell sample

The ‘Green Revolution’ dwarfing genes play a role in disease resistance in Triticum aestivum and Hordeum vulgare

The Green Revolution dwarfing genes, Rht-B1b and Rht-D1b, encode mutant forms of DELLA proteins and are present in most modern wheat varieties. DELLA proteins have been implicated in the response to biotic stress in the model plant, Arabidopsis thaliana. Using defined wheat Rht near-isogenic lines and barley Sln1 gain of function (GoF) and loss of function (LoF) lines, the role of DELLA in response to biotic stress was investigated in pathosystems representing contrasting trophic styles (biotrophic, hemibiotrophic, and necrotrophic). GoF mutant alleles in wheat and barley confer a resistance trade-off with increased susceptibility to biotrophic pathogens and increased resistance to necrotrophic pathogens whilst the converse was conferred by a LoF mutant allele. The polyploid nature of the wheat genome buffered the effect of single Rht GoF mutations relative to barley (diploid), particularly in respect of increased susceptibility to biotrophic pathogens. A role for DELLA in controlling cell death responses is proposed. Similar to Arabidopsis, a resistance trade-off to pathogens with contrasting pathogenic lifestyles has been identified in monocotyledonous cereal species. Appreciation of the pleiotropic role of DELLA in biotic stress responses in cereals has implications for plant breeding

Quantitative trait loci mapping reveals candidate pathways regulating cell cycle duration in Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Elevated parasite biomass in the human red blood cells can lead to increased malaria morbidity. The genes and mechanisms regulating growth and development of <it>Plasmodium </it><it>falciparum </it>through its erythrocytic cycle are not well understood. We previously showed that strains HB3 and Dd2 diverge in their proliferation rates, and here use quantitative trait loci mapping in 34 progeny from a cross between these parent clones along with integrative bioinformatics to identify genetic loci and candidate genes that control divergences in cell cycle duration.</p> <p>Results</p> <p>Genetic mapping of cell cycle duration revealed a four-locus genetic model, including a major genetic effect on chromosome 12, which accounts for 75% of the inherited phenotype variation. These QTL span 165 genes, the majority of which have no predicted function based on homology. We present a method to systematically prioritize candidate genes using the extensive sequence and transcriptional information available for the parent lines. Putative functions were assigned to the prioritized genes based on protein interaction networks and expression eQTL from our earlier study. DNA metabolism or antigenic variation functional categories were enriched among our prioritized candidate genes. Genes were then analyzed to determine if they interact with cyclins or other proteins known to be involved in the regulation of cell cycle.</p> <p>Conclusions</p> <p>We show that the divergent proliferation rate between a drug resistant and drug sensitive parent clone is under genetic regulation and is segregating as a complex trait in 34 progeny. We map a major locus along with additional secondary effects, and use the wealth of genome data to identify key candidate genes. Of particular interest are a nucleosome assembly protein (PFL0185c), a Zinc finger transcription factor (PFL0465c) both on chromosome 12 and a ribosomal protein L7Ae-related on chromosome 4 (PFD0960c).</p

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis

Identification and Genome-Wide Prediction of DNA Binding Specificities for the ApiAP2 Family of Regulators from the Malaria Parasite

The molecular mechanisms underlying transcriptional regulation in apicomplexan parasites remain poorly understood. Recently, the Apicomplexan AP2 (ApiAP2) family of DNA binding proteins was identified as a major class of transcriptional regulators that are found across all Apicomplexa. To gain insight into the regulatory role of these proteins in the malaria parasite, we have comprehensively surveyed the DNA-binding specificities of all 27 members of the ApiAP2 protein family from Plasmodium falciparum revealing unique binding preferences for the majority of these DNA binding proteins. In addition to high affinity primary motif interactions, we also observe interactions with secondary motifs. The ability of a number of ApiAP2 proteins to bind multiple, distinct motifs significantly increases the potential complexity of the transcriptional regulatory networks governed by the ApiAP2 family. Using these newly identified sequence motifs, we infer the trans-factors associated with previously reported plasmodial cis-elements and provide evidence that ApiAP2 proteins modulate key regulatory decisions at all stages of parasite development. Our results offer a detailed view of ApiAP2 DNA binding specificity and take the first step toward inferring comprehensive gene regulatory networks for P. falciparum

Expression of P. falciparum var Genes Involves Exchange of the Histone Variant H2A.Z at the Promoter

Plasmodium falciparum employs antigenic variation to evade the human immune response by switching the expression of different variant surface antigens encoded by the var gene family. Epigenetic mechanisms including histone modifications and sub-nuclear compartmentalization contribute to transcriptional regulation in the malaria parasite, in particular to control antigenic variation. Another mechanism of epigenetic control is the exchange of canonical histones with alternative variants to generate functionally specialized chromatin domains. Here we demonstrate that the alternative histone PfH2A.Z is associated with the epigenetic regulation of var genes. In many eukaryotic organisms the histone variant H2A.Z mediates an open chromatin structure at promoters and facilitates diverse levels of regulation, including transcriptional activation. Throughout the asexual, intraerythrocytic lifecycle of P. falciparum we found that the P. falciparum ortholog of H2A.Z (PfH2A.Z) colocalizes with histone modifications that are characteristic of transcriptionally-permissive euchromatin, but not with markers of heterochromatin. Consistent with this finding, antibodies to PfH2A.Z co-precipitate the permissive modification H3K4me3. By chromatin-immunoprecipitation we show that PfH2A.Z is enriched in nucleosomes around the transcription start site (TSS) in both transcriptionally active and silent stage-specific genes. In var genes, however, PfH2A.Z is enriched at the TSS only during active transcription in ring stage parasites. Thus, in contrast to other genes, temporal var gene regulation involves histone variant exchange at promoter nucleosomes. Sir2 histone deacetylases are important for var gene silencing and their yeast ortholog antagonises H2A.Z function in subtelomeric yeast genes. In immature P. falciparum parasites lacking Sir2A or Sir2B high var transcription levels correlate with enrichment of PfH2A.Z at the TSS. As Sir2A knock out parasites mature the var genes are silenced, but PfH2A.Z remains enriched at the TSS of var genes; in contrast, PfH2A.Z is lost from the TSS of de-repressed var genes in mature Sir2B knock out parasites. This result indicates that PfH2A.Z occupancy at the active var promoter is antagonized by PfSir2A during the intraerythrocytic life cycle. We conclude that PfH2A.Z contributes to the nucleosome architecture at promoters and is regulated dynamically in active var genes

H2A.Z Demarcates Intergenic Regions of the Plasmodium falciparum Epigenome That Are Dynamically Marked by H3K9ac and H3K4me3

Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS) that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes